Trichuris Globulosa Von Linstow, 1901 from one-humped camel (Camelus dromedarius) in Egypt: prevalence, morphological and molecular study

Background Trichuris spp. (whipworms) are soil-transmitted helminths distributed worldwide, parasitizing several mammalian hosts such as ruminants, primates, and rodents. Trichuris spp. is one of the most common intestinal parasites affecting both humans and animals, and it can spread directly through the fecal-oral route, resulting in severe illness and financial loss. So, this work aims to detect the frequency of Trichuris spp. in camels in Beheira Governorate, Egypt, and to identify Trichuris spp. through morphometrical studies, molecular analysis, and phylogenetic analysis. Results A total of 35 dromedaries out of 127 investigated had Trichuris spp. infection, meaning that the overall prevalence was 27.56%. The age of the camel affected the infection rate, older animals (> 5 years) having a higher prevalence of infection (24%) than animals of ages (< 3 years) (20%) than animals of ages (3–5 years) (19.14%). According to season: Trichuris spp. showed a unique pattern in camels in different seasons: summer (31.25%) > autumn (28.13%) > spring (25.8%) > winter (25%) indicating year-round infection. T. globulosa was identified morphometrically from camels in Beheira Governorate, Egypt. The BLAST analysis revealed the presence of T. globulosa isolate from camels using the Genbank database depending on nuclear small subunit ribosomal RNA (18s) and cytochrome b (Cytb) genes. Conclusion A high prevalence of T. globulosa was found in camels in Beheira Governorate, Egypt. This is the first report to confirm the identification of T. globulosa from camel based on morphometrical studies and molecular and phylogenetic analysis in Egypt. More thorough studies on the incidence, molecular, and genetic analysis of Trichuris spp. in Egypt are required in addition to camel control programs.


Background
Throughout the world, one-humped camels (Camelus dromedarius) serve various purposes.Many parts of the world use camels as a source of milk, meat, and transportation [1,2].
Infected camels with parasites produced less milk and meat, had poorer fertility, and had lower calving rates [4].Camels play a significant role in the epidemiology of parasitic diseases [5].In Egypt, the camel population was estimated at 80,000, most of which was in upper Egypt.
Helminths that could infect camels include trematodes, cestodes, and nematodes.Whipworms are soil-transmitted nematodes distributed worldwide, parasitizing several mammalian hosts such as ruminants, primates, and rodents [6,7].Trichuris spp. is one of the most common intestinal parasites affecting both humans and animals, and it can spread directly through the fecal-oral route, resulting in severe illness and financial loss [8,9].
Trichuris is closely related to Trichinella and Capillaria and belongs to the class Enoplea, subclass Dorylaimia, and order Trichinellida [10].The diagnosis of Trichuris is made by observing eggs in fecal samples in living animals [11].
As many characters used to distinguish between species interfere, Trichuris species morphological identification is difficult [12].The spicule length is the most essential character to differentiate Trichuris spp [13].
In the distal portion of the spicule sheath of T. globulosa, males exhibit a spherical bulge covered with longer spines than the rest of the sheath.Less than 4.9 mm in spicule length and no prominent vulva in females [13,14].
Until now, there have been few studies on T. globulosa in camels, especially in Egypt.So, this work aims to identify the prevalence of T. globulosa in camels in Beheira Governorate, Egypt, through morphometrical studies, molecular analysis, and phylogenetic analysis.

Samples
Each cecum was ligated and removed from the camels slaughtered at the abattoir, collected in clean bags labeled (age, seasonal dynamics), and transferred to the laboratory of Parasitology in an ice container for further examinations.Initial examination was done within hours (24 h.) after transportation.

Parasitic materials
In the laboratory of the parasitology Department, Faculty of Veterinary Medicine, Kafrelsheikh University, Egypt, each cecum was opened; contents were evacuated and scrutinized for whipworms with the aid of a hand magnification lens and OLYMPUS stereomicroscope.The worms were collected from the collected cecum.The worms were preserved in formalin 10% and ethyl 70% alcohol for morphometrical identification and molecular analysis.

Molecular examination
Genomic DNA was extracted from three adult Trichuris spp.samples using the QIAamp DNA Mini Kit (Catalogue no.51,304) following the instructions of the manufacturing.

Molecular characterization
The genes for cytochrome b (Ctyb) and Nuclear small subunit ribosomal RNA (18s rRNA) were subjected to amplification.Fragments of genes amplified using specific primers. (

-G A G T A A T T T T T A T A A T A C G A G A A G T-3 ) and (-A A T T T T C A G G G T C T C T G C T T C A A T A-3
) for Cytb as forward and reverse primers, respectively [11]. ( as forward and reverse primers, respectively [21].
Cycling conditions and PCR Mix were applied as Callejón et al. [15] and Floyd et al. [21].
Electrophoresis with 1.5% Agarose gel in TAE buffer and stained with 0.5 µg/ml ethidium bromide (Sigma) was used for visualization of PCR products.A 100-bp ladder and, a known sample (positive control), and distilled water (negative control) were included in the gel.A gel documentation system (SYNGENE) was used to take images of the gels, and computer software was used to analyze the data.

Sequencing and phylogenetic study
Using an Applied Biosystems 3130 automated DNA sequencer (ABI, 3130, USA), a purified PCR product of the Cytb and 18s genes was sequenced forward and backward.Using a ready reaction Bigdye Terminator V3.1 cycle sequencing kit.(Perkin-Elmer/Applied Biosystems, Foster City, CA).
A BLAST analysis (Basic Local Alignment Search Tool) was initially performed to establish sequence identity to GenBank accessions [22].Higher identity sequences were downloaded from Genbank, and Trichinella spiralis was used as an out-group species for phylogenesis.Sequences were aligned using the alignment tool in Mega 11 software [23].
A phylogenetic tree was built using the Maximum Likelihood method and the Tamura Nei model [24].
Finally, the sequences obtained in this work were submitted to Genbank for accession numbers.

Statistical analysis
A statistical application called GraphPad Prism 9 was used to examine statistical significance differences using a Chi-square.P values less than 0.05 were used to determine statistical significance.

Survey finding
A total of 35 dromedaries out of 127 investigated had Trichuris spp.infection, meaning that the overall prevalence was 27.56%.
The age of the camel affected the infection rate, older animals (> 5 years) having a higher prevalence of infection (24%) than animals of ages (< 3 years) (20%) than animals of ages (3-5 years) (19.14%).However, the statistical analysis indicated these changes were not statistically significant.

Morphological and biometrical results (Fig. 2)
We studied the morphology of thirty adult camel worms (fifteen male and fifteen female) isolated from the camel's cecum.The vaginal length of females varied from 0.7 to 0.9 mm with an average: 0.79 mm, while their overall length varied from 37.7 to 59.9 mm (average: 52.9 mm).A morphological analysis revealed that the vulva had a round margin free from spines.Vulva looked smooth.A tiny egg chamber was located anterior to the vulva, and the vagina was short and occasionally slightly convoluted in multiple loops.
Males had a total length of 43.5 to 62.8 mm (average: 53.3 mm), while the spicule length ranged from 3.91 to 4.9 mm (average: 4.4 mm).Spicule diameters varied from 0.031 to 0.075 mm (average of 0.046 mm).The distal portion of the spicule sheath in males featured a spherical bulge covered in spines that were longer than the remaining spines in the sheath.

Molecular results
Using particular primers, the cytb and18s genes were amplified by PCR to produce bands measuring approximately 450 bp and 900 bp, respectively.The adult T. globulosa Ctyb sequences were placed in Genbank with accession numbers OR863681, OR863682, and OR863683 and varied between 439 and 444 bp.while the adult T. globulosa 18s sequences placed in Genbank with accession numbers OR775092, OR775093, and OR775096 and varied from 786 to 860 bp.The Genbank database was used for the BLAST analysis of the sequenced data, which indicated the existence of the T. globulosa isolate from camels based on the 18s and Cytb genes.
This study identified Trichuris from Egypt as T. globulosa based on morphological and molecular examinations.

Discussion
Important camelid parasites, whipworms (Trichuris spp.) are under-estimated parasite infections compared with other gastrointestinal nematodes, although they could result in a significant loss.The large intestine and caecum contain adult parasites.Significant enteritis brought on by Trichuris spp.causes diarrhea, dehydration, and weight loss [25].The most common and widespread trichurid in camels is Trichuris globulosa [26].According to the current study, Trichuris spp. was present in 27.56% of the camels in Beheira that had been slaughtered.The findings of the current study were lower than 40% in Iran [27] and higher than the1.5%,4.1%, 4.9%and 12.24% in Pakistan [28], Iran [29,30], and Toukh, Egypt [31], respectively.Variations in ecological location, conditions of the environment, animal rearing practices, degree of exposure to infectious eggs, and hygienic system could all contribute to the variation in prevalence.
In this study, Trichuris globulosa was detected at all ages, and the infection rates at ages of (< 3 years), (3-5 years) and (> 5 years) were 20%, 19.14%, and 24%.These results were matched those of the previous study [31].This could be because the older animals have contact with the eggs of Trichuris spp.more than young animals.
Summer and spring had the highest infection rates, followed by autumn and then winter.Some studies showed the infection rate was the highest in winter [31].Differences in environmental conditions, pastures among seasons, and geographical distribution could cause these variations.
The structure and lining of the vagina and the distance between the vulva and the uterine sphincter are the main factors that determine how differentiable the females of Trichuris spp.are from one another.The distance between the vulva and uterine sphincter of the females of T. globulosa of Iranian camels varied from 0.7 to 0.8 mm (average: 0.73 mm) [15] in contrast, it varied from 0.7 to 0.9 mm (average: 0.79 mm) in the current study.For T. discolor, this distance varied from 3.6 to 5.3 mm [34] and 1.40 to 2.85 mm [36], while T. tenuis varied from 2.5 to 3.4 mm [36].
The spicule length was considered the primary characteristic distinguishing Trichuris species [13,15].
In line with T. globulosa, the male Trichuris in the current study on Egyptian camels displayed a range of 4.1 mm to 5.1 mm [13][14][15]38] but slightly overlapping T. ovis.
The distal end of the spicule of males of Trichuris from Egypt is pointed, resembling T. globulosa, whereas the distal end of the spicule in T. tenuis and T. discolor is bluntly rounded [13][14][15]35].
Although there are some notable differences between T. globulosa and T. ovis, these two species are remarkably alike in size and external body shape as well as the size and shape of the eggs (the spicule lengths of 2 species overlapped slightly) [13].Specifically, the males of both species have a spherical bulge at the distal end of the spicule sheath when fully protruded.T. globulosa exhibits noticeably longer protrusion spines, while the protrusion's spines in T. ovis are smaller than those on the rest of the sheath.Overall, spicule sheath spines are generally longer and closer together in T. globulosa than those in T. ovis [14,39,40].Tenora et al. [38] disagreed with these studies and concluded that there isn't a difference in character between the two species.
The nuclear small subunit ribosomal RNA (18s rRNA) gene has been widely employed in nematode phylogenetics.This gene was sequenced and used in phylogenetics in more than 1000 species of nematodes [16,17].
According to the mitochondrial data, there is a different genetic lineage between T. ovis and T. globulosa.A distinct genetic lineage of T. ovis from South African sheep, which would be closely linked to the T. globulosa populations found in Iranian camels, was confirmed by cytochrome b partial gene sequences (Ctyb).For the first time, the cytb partial gene sequences of T. globulosa have been published [15].Through BLAST, a comparison of the newly obtained Cytb and 18s sequence with other Trichuris sequences on Genbank, the current study's sequence indicated that the species was T. globulosa.
A phylogenetic tree based on Cytb revealed high similarity between Trichuris globulosa in the present study and T. globulosa from camels in Iran [15], which reported T. globulosa's Cytb partial gene sequences for the first time, and this study reported T. globulosa's 18s partial gene sequences for the first time.

Conclusion
A high prevalence of T. globulosa was found in camels in Beheira Governorate, Egypt.This is the first report to confirm the identification of T. globulosa from camel based on morphometrical studies and molecular and phylogenetic analysis in Egypt.thorough studies on the incidence, molecular, and genetic analysis of Trichuris spp. in Egypt are required in addition to camel control programs.

Fig. 1
Fig. 1 Map of Egypt, illustrating the sampling site

Fig. 2
Fig. 2 Trichuris globulosa detected in the current study: (a) and (b) female vulva and vagina, (c) and (d) egg (scale bar 70 μm), (e) male caudal region showing the spicule, the evaginated spicule sheath and (f) spherical bulge covered with spines

Fig. 4 Fig. 3
Fig. 4 Maximum Likelihood tree based on 18s partial gene of ribosomal RNA gene of Trichuris globulosa in the current study, Trichinella spiralis was used as the outgroup.* Current study Fig. 3 Maximum Likelihood tree based on Cytb DNA gene of Trichuris globulosa in the current study, Trichinella spiralis was used as the outgroup.* Current study

Table 1
Trichuris globulosa measurements (mm) from current study with comparison with previous studies